The utility of many biologic drugs is limited by inefficient cellular delivery [1]. Additional problems include biological instability, poor biodegradability or immunogenicity [1e]. Previous efforts to overcome this limitation have focused largely on the use of cationic domains, including peptidic cationic species (e.g., HIV-TAT, penetratin, and nonaarginine and more generally cell penetrating peptides (CPP)) or non-peptidic cationic species (e.g., PAMAM dendrimers and polyethylimine), to enhance the attraction between a chemotherapeutic agent and the anionic cell surface [2]. Natural ligands (e.g., folic acid, substance P, and the RGD tripeptide) have also been used to facilitate cellular delivery by targeting agents to specific cell-surface receptors [3]. Such methods have been applied, for example, to delivery of peptides, proteins, nucleic acids and analogs thereof, reporters and labels, various pharmaceuticals and drugs and various small molecules as well as particles. Although some of these methods have had some success, there remains a need in the art for additional delivery strategies.
The cell surface of many prokaryotic and eukaryotic cells is coated with a dense forest of polysaccharides known as the glycocalyx [4]. Targeting of therapeutic agents to the glycocalyx might enhance their cellular delivery, as has been demonstrated with lectin conjugates [5]. Boronic acids readily form boronate esters with the 1,2- and 1,3-diols of saccharides [6], including those in the glycocalyx [7]. In addition, boronate groups are compatible with human physiology, appearing in chemotherapeutic agents and other remedies [8]. The present invention relates to the use of pendant boronic acids to mediate the delivery of a cargo molecule into mammalian cells.
Boronic acids have been employed in the development of sensors for glycans [6] and in the development of boron neutron capture therapies for cancer treatment [24]. Boronic acids have also been reported as useful in glucose sensors for the controlled release of insulin [8d]. Certain diboronic acid compounds have been reported useful as fluorescent sensors for certain cancer cells which express sialyl Lewis X [7b]. Boronic acids have been reported useful in drug delivery applications, for example, to transport nucleotides and monosaccharides across liposomal bilayers [8a, 25], for labeling of liposomes for increased affinity for erythrocyte ghosts [7a] and for enhancement of PEI-DNA complexation for transfection reagents [8e]. Phenylboronic acid-(PBA-) functionalized polymers [26, 27] and nanoparticle complexes [28, 29, 30] have been loaded with small-molecule chemotherapeutic agents. These approaches employ large, complex assemblies that use PBA as a reactive functionality [31] or in the architecture of the delivery complex 32-36]. PBA has also been expanded to deliver protein cargos under conditions of oxidative stress [34] and to deliver DNA. [8e, 37]
U.S. Pat. No. 4,499,082 reports certain alpha-aminoboronic acid peptides as reversible inhibitors of proteolytic enzymes. U.S. Pat. No. 6,018,020 relates to boronated amino acid derivatives which are viral proteinase inhibitors. U.S. Pat. Nos. 6,465,433; 6,548,668; and U.S. published application 2009/0325903 report certain amino acid and peptidyl boronic acids as inhibitors of proteasome function and more specifically as inhibitors of HIV replication in an animal. In each of these references, a carboxylic acid group of an amino acid is replaced with a boronic acid group (—B(OH)2) or an ester thereof. Each of these references is incorporated by reference herein in its entirety for its description of amino acids and peptidyl boronic acids and any description therein of methods of synthesis thereof.
U.S. published application 20110059040 relates to methods for inhibiting the activity of a virus or bacterium by contacting the virus or bacterium with a polymer functionalized with at least one boronic acid moiety, including one or more substituted or unsubstituted arylboronic acids, particularly phenylboronic acids. Certain multivalent benzoboroxole functionalized polymers have been reported to inhibit microbicide entry [8b].
U.S. Pat. Nos. 5,594,111; 5,594,151; 5,623,055; 5,777,148; 5,744,627; 5,837,878; and 6,156,884 relate to phenylboronic acid complexing reagents and complexes formed with such complexing reagents which are reported to be useful for preparation of bioconjugates. Similar reports of phenyldiboronic acid complexing reagents and complexes formed therewith are found in U.S. Pat. Nos. 6,075,126; 6,124,471; 6,462,179 and 6,630,577. Each of these references is incorporated by reference herein in its entirety for descriptions of phenylboronic acid compounds and phenyldiboronic acid compounds and methods of synthesis thereof.
The physical properties including sugar binding properties of certain benzoboroxoles and benzoxaborins have been studied [13, 22, 27]. Syntheses of certain benzoboroxoles have been reported [30-36]. A review of applications of benzoboroxoles has been provided [28]. These references are incorporated by reference herein for methods of synthesis and sugar binding properties of benzoboroxoles.
The design, synthesis and screening of a library of peptidyl bis(boroxoles) as receptors for the tumor marker TF-Antigen disaccharide in water has been reported [22]. The library components contained a free amine, a certain PEG linker, two diaminoproponic acids residues to which benzoboroxole groups were attached separated by a central amino acid (where the library components were randomized with 20 selected natural and non-natural amino acids) and a capping group (where the library components were randomized with 20 selected capping groups). This reference is incorporated by reference herein for its description of the library species and methods of synthesis of the library species. Any peptidyl bis(boroxoles) noted in this reference can optionally be excluded from the claims herein.
Gold electrodes activated by phenylboronic acid diazonium salts are reported for the reversible immobilization of eukaryotic cells [7c]. An electrochemical cell sensor for the determination of K562 leukemia cells using 3-aminophenylboronic acid (APBA)-functionalized multiwalled carbon nanotubes (MWCNTs) films is reported [7e]. K562 leukemia cells are reported to be bound to the APBA-functionalized MWCNTs film via boronic acid groups. This reference is incorporated by reference herein in its entirety for descriptions of phenylboronic acid diazonium salts and synthesis thereof.